The long-term goal of this research project is the elucidation of the mechanisms of protective immunity against Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis (HME). Achievement of this goal requires knowledge of the ehrlichial antigens that stimulate protective immunity and of the humoral and cellular immune mechanisms that are effective in the clearance of ehrlichiae from the infected macrophages throughout the body. HME is a life-threatening tick-borne infection that has been associated with toxic shock syndrome-like manifestations, adult respiratory distress syndrome, meningitis, and disseminated intravascular coagulation in immunocompetent patients, overwhelming opportunistic infection in immunocompromised patients, and a fatality rate of 2.7%. The specific aims, designed to test the hypothesis that the immunodominant, surface-exposed p 120 and p28-family stimulate protective immunity by a combination of antibodies and cellular mechanisms, are 1) to determine the roles of proteins of the p28 family and pl2O in stimulating protective immunity in a mouse model of HME against an organism closely related to E. chaffeensis, an Ehrlichia species isolated from Japanese Ixodes ovatus ticks (IOE); 2) to determine the importance and mechanism(s) of antibodies in protective immunity against IOE in the mouse model of HME; and 3) to identify the cellular immune mechanisms that are critical in protective immunity against IOE in the mouse model of HME. The research design includes purification of recombinant pl2O and each member of the p28 family of IOE, production of DNA vaccines expressing each of these proteins, and testing the DNA and recombinant protein vaccines in the highly pathogenic IOE C57BL/6 mouse model. The hypothesis of humorally mediated immunity will be examined by passive immunization studies with specific polyclonal and monoclonal antibodies to p I 2O and the p28 family in IOE-challenged immunocompetent, SCID, and Fc-receptor knockout mice. Opsonization will be investigated in murine and human macrophages in vitro with E. chaffeensis and specific polyclonal and monoclonal antibodies. Cellular immune mechanisms will be elucidated using gene knockout mice (MHC Class I, MHC Class II, 8 T-cell receptor, IFN-Y, perforin, INOS), immunodeficient SCID and Beige, and TNF-alpha depleted mice, immunohistochemical and flow cytometric analyses of the cell subsets and their cytokine profiles, adoptive transfer of T-lymphocytes and their subsets including antigen-specific T-cell lines, and determination of the chemokines that are important for immunity to the IOE in an outstanding new mouse model of HME.